Methods for preparing polymeric reagents and compositions of polymeric reagents

ABSTRACT

Methods for preparing active carbonate esters of water-soluble polymers are provided. Also provided are other methods related to the active carbonate esters of water-soluble polymers, as well as corresponding compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/497,463, filed Jul. 31, 2006, now U.S. Pat. Ser. No. 7,767,784, whichapplication claims the benefit of priority to U.S. Provisional patentapplication Ser. No. 60/703,709, filed Jul. 29, 2005, the contents ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to methods for preparing water soluble andnon-peptidic polymers (“polymeric reagents”) as well as to compositionsof water-soluble of the same, conjugates, pharmaceutical compositions,and methods of administering pharmaceutical compositions.

BACKGROUND OF THE INVENTION

Scientists and clinicians face a number of challenges in their attemptsto develop active agents into forms suited for delivery to a patient.Active agents that are proteins, for example, are often delivered viainjection rather than orally. In this way, the protein is introducedinto the systemic circulation without exposure to the proteolyticenvironment of the stomach. Injection of proteins, however, has severaldrawbacks. For example, many proteins have a relatively short half-life,thereby necessitating repeated injections, which are often inconvenientand painful. Moreover, some proteins may elicit one or more immuneresponses with the consequence that the patient's immune system attemptsto destroy or otherwise neutralize the immunogenic protein. Of course,once the protein has been destroyed or otherwise neutralized, theprotein cannot exert its intended pharmacodynamic activity. Thus,delivery of active agents such as proteins is often problematic evenwhen these agents are administered by injection.

Some success has been achieved in addressing the problems of deliveringactive agents via injection. For example, conjugating an active agent toa water-soluble polymer has resulted in a polymer-active agent conjugatehaving reduced immunogenicity and antigenicity. In addition, thepolymer-active agent conjugate often has an increased half-life comparedto its unconjugated counterpart as a result of decreased clearancethrough the kidney and/or decreased enzymatic degradation in thesystemic circulation. As a result of having a greater half-life, thepolymer-active agent conjugate requires less frequent dosing, which inturn reduces the overall number of painful injections and inconvenientvisits with a health care professional. Moreover, active agents thatwere only marginally soluble demonstrate a significant increase in watersolubility when conjugated to a water-soluble polymer.

Due to its documented safety as well as its approval by the FDA for bothtopical and internal use, polyethylene glycol has been conjugated toactive agents. When an active agent is conjugated to a polymer ofpolyethylene glycol or “PEG,” the conjugated active agent isconventionally referred to as “PEGylated.” The commercial success ofPEGylated active agents such as PEGASYS® PEGylated interferon alpha-2a(Hoffmann-La Roche, Nutley, N.J.), PEG-INTRON® PEGylated interferonalpha-2b (Schering Corp., Kennilworth, N.J.), and NEULASTA™PEG-filgrastim (Amgen Inc., Thousand Oaks, Calif.) demonstrates thatadministration of a conjugated form of an active agent can havesignificant advantages over the unconjugated counterpart. Smallmolecules such as distearoylphosphatidylethanolamine (Zalipsky (1993)Bioconjug. Chem. 4(4):296-299) and fluorouracil (Ouchi et al. (1992)Drug Des. Discov. 9(1):93-105) have also been PEGylated. Harris et al.have provided a review of the effects of PEGylation on pharmaceuticals.Harris et al. (2003) Nat. Rev. Drug Discov. 2(3):214-221.

Typically, the formation of a conjugate involves reaction between anactive agent and a polymeric reagent. While small scale amounts ofpolymeric reagents are available from commercial sources such as NektarTherapeutics, a concern arises when a commercial or production scale ofthe polymeric reagent is required. In particular, there is a concernthat the particular polymeric reagent used in making the desiredconjugate (or an intermediate useful in preparing the polymeric reagentused in making the desired conjugate) cannot be synthesized, largelyfree of potentially harmful impurities, in a timely, efficient, andeconomical manner.

For example, the conventional synthesis of polymers bearing an activeester—which can be used a polymeric reagent as well as an intermediateuseful in preparing other polymeric reagents—requires an excess of a lowmolecular weight reagent [such as di(1-benzotriazolyl) carbonate], whichmust be removed. Although complicated, removal of the low molecularweight reagent is necessary so that the low molecular weight reagentdoes not react with other molecules, thereby introducing undesired sidereactions and products that result in a relatively impure product anddecreased yield.

In one approach for preparing polymers bearing an active ester, U.S.Pat. No. 6,624,246 describes the synthesis of methoxy poly(ethyleneglycol) bearing a benzotriazole carbonate group (“mPEG-BTC”). Asdescribed therein, the process effectively involves mPEG-BTC formationfollowed by mPEG-BTC purification to purify the mPEG-BTC species andremove unreacted di(1-benzotriazolyl) carbonate and any other lowmolecular weight products (e.g., 1-benzotriazolyl alcohol). As describedin U.S. Pat. No. 6,624,246, the purification of mPEG-BTC involvesmultiple precipitation steps. A schematic of the process is providedbelow.

-   -   B) mPEG-BTC Purification        -   a) distill off the organic solvent to form a residue of the            three products        -   b) re-dissolve the three products in methylene chloride        -   c) add ethyl ether, cool and form a precipitate of

In the manufacture of mPEG-BTC wherein the poly(ethylene glycol) portionhas a weight-average molecular weight of about 20,000 Daltons, aneight-fold excess of diBTC was used in order to achieve 100% conversionof all mPEG-OH to mPEG-BTC. Although the large amount of diBTC ensuresoptimal conversion to MPEG-OH to mPEG-BTC, a relatively large amount ofdiBTC remains unreacted and must be removed prior to carrying out anyfurther synthetic steps. Otherwise, the remaining diBTC would react withany reactive group (e.g., alcohol group, amine group, and so forth)encountered and introduce undesired impurities and reduce the overallyield.

As described in U.S. Pat. No. 5,932,462, mPEG-BTC was reacted withlysine (bearing two amino groups and a single carboxylic acid group),thereby providing a “lysine branched” structure wherein an mPEG residueis attached at each of the amino groups and the carboxylic acid isavailable for further functionalizing. While it is conceivable toconsume any excess diBTC with adding an excess of lysine, such anapproach is flawed for at least two reasons. First, both the diBTC andmPEG-BTC will “compete” for the available lysine amino groups, therebyresulting in a mixture of the desired lysine branched structure andanother species having only a single mPEG residue, a single BTC group,and a single carboxylic acid. Second, even if this approach wassuccessful, it would not address situations where a non-lysineresidue-containing product is desired.

One may avoid having excess diBTC during the lysine reaction only bydestroying the excess diBTC from formation of mPEG-BTC during mPEG-BTCpurification. Isopropyl alcohol (IPA, isopropanol) may be substitutedfor ethyl ether to precipitate the mPEG-BTC from a methylene chloridesolution [i.e., e.g., step B(c) in the above schematic]. If this changeis made, the IPA reacts with the excess diBTC to form isopropyl-BTC,which is soluble in the mixture of methylene chloride and IPA.Unfortunately, in such an operation at large scale, some isopropyl-BTCis trapped in the mPEG-BTC precipitate. Since the isopropyl-BTC wouldcompete with mPEG-BTC in any reaction with lysine, the isopropyl-BTCmust be removed before manufacturing can continue. To remove the trappedisopropyl-BTC, one or two additional “re-precipitation” steps must becarried out in order to get mPEG-BTC free of isopropyl-BTC. As eachre-precipitation gives some loss of the mPEG-BTC product because onlyabout 85-95% of the solid can be recovered, this approach is costly andrequires additional time.

In another approach for preparing polymers bearing an active ester, U.S.Pat. No. 5,281,698 describes the synthesis of methoxy poly(ethyleneglycol) bearing a succinimide carbonate group (“mPEG-SC”). As describedtherein, the process effectively involves mPEG-SC formation followed bymPEG-SC purification to purify the mPEG-SC species and remove unreacteddisuccinimidyl carbonate and any other low molecular weight products(e.g., N-hydroxysuccinimide). As described in U.S. Pat. No. 5,281,698,the purification of mPEG-SC involves filtration and multipleprecipitation steps. A schematic of the process is provided below.

-   -   A) mPEG-SC Formation

-   -   B) mPEG-SC Purification        -   a) filter the reaction mixture        -   b) distill off the organic solvent to form a residue of the            three products        -   c) re-dissolve the three products in methylene chloride        -   d) add ethyl ether, cool and form a precipitate of mPEG-SC        -   f) repeate precipitation (step c and d) two more times

In the manufacture of mPEG-SC wherein the poly(ethylene glycol) portionhas a weight-average molecular weight of about 6100 Daltons, antwenty-fold excess of DSC was used in order to achieve 100% conversionof all mPEG-OH to mPEG-SC. Although the large amount of DSC ensuresoptimal conversion to MPEG-OH to mPEG-SC, a relatively large amount ofDSC remains unreacted and must be removed prior to carrying out anyfurther synthetic steps. Otherwise, the remaining DSC would react withany reactive group (e.g., alcohol group, amine group, and so forth)encountered and introduce undesired impurities and reduce the overallyield.

Thus, there remains a need for an efficient method to remove excess lowmolecular weight reagents, such as diBTC and/or its reactive degradants,from the same reaction mixture in which the low molecular weight reagentwas added, thereby resulting in a “one-pot” reaction. This presentinvention addresses this and other needs in the art.

SUMMARY OF THE INVENTION

In one or more embodiments, a synthetic method is provided, the methodcomprising:

(a) combining a composition comprising an amine-terminated orhydroxy-terminated, water-soluble polymer with a composition comprisingan activated carbonate reagent, optionally in the presence of a catalystor acid-neutralizing base, wherein the composition comprising theactivated carbonate reagent is added such that there is an excess of theactivated carbonate reagent relative to the amine-terminated orhydroxy-terminated, water-soluble polymer, to thereby result in acomposition comprising an active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent; and

(b) adding a composition comprising a reactive molecule to thecomposition comprising the active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent, wherein thecomposition comprising a reactive molecule is added such thatsubstantially all of the unreacted activated carbonate reagent issubstantially consumed.

In one or more embodiments of invention, a synthetic method is provided,the method comprising:

(a) combining a composition comprising a hydroxy-terminated,water-soluble polymer having the following structure:CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OHwherein (n) is an integer from 2 to about 4000, with a compositioncomprising di(1-benzotriazolyl) carbonate, wherein the compositioncomprising the di(1-benzotriazolyl) carbonate is added such that thereis an excess of the di(1-benzotriazolyl) carbonate relative to thehydroxy-terminated, water-soluble polymer, to thereby result in acomposition comprising an active carbonate ester of the water-solublepolymer having the following structure:

wherein (n) is an integer from 2 to about 4000, and unreacteddi(1-benzotriazolyl) carbonate;

-   (b) adding a composition comprising a reactive molecule to the    composition comprising the active carbonate ester of a water-soluble    polymer and unreacted di(1-benzotriazolyl) carbonate, wherein the    composition comprising the reactive compound is added such that    substantially all of the unreacted di(1-benzotriazolyl) carbonate is    substantially consumed.

In one or more embodiments of the invention, a synthetic method isprovided, the method comprising:

(a) combining a composition comprising a hydroxy-terminated,water-soluble polymer having the following structure:CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OHwherein (n) is an integer from 2 to about 4000, with a compositioncomprising disuccinimidyl carbonate, wherein the composition comprisingthe disuccinimidyl carbonate is added such that there is an excess ofthe disuccinimidyl carbonate relative to the hydroxy-terminated,water-soluble polymer, to thereby result in a composition comprising anactive carbonate ester of the water-soluble polymer having the followingstructure:

wherein (n) is an integer from 2 to about 4000, and unreacteddisuccinimidyl carbonate;

(b) adding a composition comprising a reactive molecule to thecomposition comprising the active carbonate ester of the water-solublepolymer and unreacted disuccinimidyl carbonate, wherein the compositioncomprising the reactive molecule is added such that substantially all ofthe unreacted disuccinimidyl carbonate is substantially consumed.

In one or more embodiments of the invention, a synthetic method isprovided, the method comprising:

(a) combining a composition comprising a hydroxy-terminated,water-soluble polymer having the following structure:CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OHwherein (n) is an integer from 2 to about 4000, with a compositioncomprising p-nitrophenyl chloroformate, wherein the compositioncomprising the p-nitrophenyl chloroformate is added such that there isan excess of the p-nitrophenyl chloroformate relative to thehydroxy-terminated, water-soluble polymer, to thereby result in acomposition comprising an active carbonate ester of the water-solublepolymer having the following structure:

wherein (n) is an integer from 2 to about 4000, and unreactedp-nitrophenyl chloroformate;

(b) adding a composition comprising a reactive molecule to thecomposition comprising the active carbonate ester of water-solublepolymer and unreacted p-nitrophenyl chloroformate,

wherein the composition comprising the reactive molecule is added suchthat substantially all of the unreacted p-nitrophenyl chloroformate issubstantially consumed.

In one or more embodiments of the invention, a synthetic method isprovided, the method comprising:

-   -   reacting an active carbonate ester of a water-soluble polymer        prepared by

(i) combining a composition comprising an amine-terminated orhydroxy-terminated, water-soluble polymer with a composition comprisingan activated carbonate reagent, optionally in the presence of a catalystor acid-neutralizing base, wherein the composition comprising theactivated carbonate reagent is added such that there is an excess of theactivated carbonate reagent relative to the amine-terminated orhydroxy-terminated, water-soluble polymer, to thereby result in acomposition comprising an active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent; and

(ii) adding a composition comprising a reactive molecule to thecomposition comprising the active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent, wherein thecomposition comprising a reactive molecule is added such thatsubstantially all of the unreacted activated carbonate reagent issubstantially consumed,

-   -   with an active agent under conjugation conditions to thereby        result in a water-soluble polymer-active agent conjugate.

In one or more reacting a polymeric reagent prepared by

(a) combining a composition comprising an amine-terminated orhydroxy-terminated, water-soluble polymer with a composition comprisingan activated carbonate reagent, optionally in the presence of a catalystor acid-neutralizing base, wherein the composition comprising theactivated carbonate reagent is added such that there is an excess of theactivated carbonate reagent relative to the amine-terminated orhydroxy-terminated, water-soluble polymer, to thereby result in acomposition comprising an active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent;

(b) adding a composition comprising a reactive molecule to thecomposition comprising the active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent, wherein thecomposition comprising a reactive molecule is added such thatsubstantially all of the unreacted activated carbonate reagent issubstantially consumed; and

(c) reacting the active carbonate ester of the water-soluble polymer inone or more reactions to form a polymeric reagent

with an active agent under conjugation conditions to thereby result in awater-soluble polymer-active agent conjugate.

In one or more embodiments of the invention, a synthetic method isprovided, the method comprising:

reacting a polymeric reagent prepared by

(a) combining a composition comprising an amine-terminated orhydroxy-terminated, water-soluble polymer with a composition comprisingan activated carbonate reagent, optionally in the presence of a catalystor acid-neutralizing base, wherein the composition comprising theactivated carbonate reagent is added such that there is an excess of theactivated carbonate reagent relative to the amine-terminated orhydroxy-terminated, water-soluble polymer, to thereby result in acomposition comprising an active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent;

(b) adding a composition comprising a reactive molecule to thecomposition comprising the active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent, wherein thecomposition comprising a reactive molecule is added such thatsubstantially all of the unreacted activated carbonate reagent issubstantially consumed; and

(c) reacting the active carbonate ester of the water-soluble polymer inone or more reactions to form a polymeric reagent

with an active agent under conjugation conditions to thereby result in awater-soluble polymer-active agent conjugate.

In one or more embodiments of the invention, a method for preparing aconjugate-containing composition is provided, the method comprisingcombining an active carbonate ester of a water soluble polymer or(polymeric reagent prepared therefrom) as prepared according to asynthetic method described herein with an active agent to thereby resultin a conjugate-containing composition.

In one or more embodiments of the invention, a conjugate-containingcomposition is provided, the composition resulting from the method for,preparing a conjugate-containing composition as provided herein.

DETAILED DESCRIPTION OF THE INVENTION

Before describing one or more embodiments of the present invention indetail, it is to be understood that this invention is not limited to theparticular polymers, reagents, and the like, as such may vary.

It must be noted that, as used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “apolymer” includes a single polymer as well as two or more of the same ordifferent polymers, reference to “an activated carbonate reagent” refersto a single activated carbonate reagent as well as two or more of thesame or different activated carbonate reactive agents, and the like.

In describing and claiming the present invention(s), the followingterminology will be used in accordance with the definitions providedbelow.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are interchangeable. Typically, PEGs for use in accordance with theinvention comprise the following structure: “—(OCH₂CH₂)_(n)—” where (n)is 2 to 4000. As used herein, PEG also includes“—CH₂CH₂—O(CH₂CH₂O)_(n)—CH₂CH₂—” and “—(OCH₂CH₂)_(n)O—,” depending uponwhether or not the terminal oxygens have been displaced. Throughout thespecification and claims, it should be remembered that the term “PEG”includes structures having various terminal or “end capping” groups. Theterm “PEG” also means a polymer that contains a majority, that is tosay, greater than 50%, of —OCH₂CH₂— or —CH₂CH₂O-repeating subunits. Withrespect to specific forms, the PEG can take any number of a variety ofmolecular weights, as well as structures or geometries such as“branched,” “linear,” “forked,” “multifunctional,” and the like, to bedescribed in greater detail below.

The terms “end-capped” and “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group, more preferably aC₁₋₁₀ alkoxy group, and still more preferably a C₁₋₅ alkoxy group. Thus,examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxyand benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. It must be remembered that the end-capping moiety may include oneor more atoms of the terminal monomer in the polymer [e.g., theend-capping moiety “methoxy” in CH₃(OCH₂CH₂)_(n)—]. In addition,saturated, unsaturated, substituted and unsubstituted forms of each ofthe foregoing are envisioned. Moreover, the end-capping group can alsobe a silane. The end-capping group can also advantageously comprise adetectable label. When the polymer has an end-capping group comprising adetectable label, the amount or location of the polymer and/or themoiety (e.g., active agent) to which the polymer is coupled can bedetermined by using a suitable detector. Such labels include, withoutlimitation, fluorescers, chemiluminescers, moieties used in enzymelabeling, calorimetric moieties (e.g., dyes), metal ions, radioactivemoieties, and the like. Suitable detectors include photometers, films,spectrometers, and the like. The end-capping group can alsoadvantageously comprise a phospholipid. When the polymer has anend-capping group comprising a phospholipid, unique properties areimparted to the polymer and the resulting conjugate. Exemplaryphospholipids include, without limitation, those selected from the classof phospholipids called phosphatidylcholines. Specific phospholipidsinclude, without limitation, those selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin.

“Non-naturally occurring” with respect to a polymer as described herein,means a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer may, however, contain one or moremonomers or segments of monomers that are naturally occurring, so longas the overall polymer structure is not found in nature.

The term “water soluble” as in a “water-soluble polymer” is any polymerthat is soluble in water at room temperature. Typically, a water-solublepolymer will transmit at least about 75%, more preferably at least about95%, of light transmitted by the same solution after filtering. On aweight basis, a water-soluble polymer will preferably be at least about35% (by weight) soluble in water, more preferably at least about 50% (byweight) soluble in water, still more preferably about 70% (by weight)soluble in water, and still more preferably about 85% (by weight)soluble in water. It is most preferred, however, that the water-solublepolymer is about 95% (by weight) soluble in water or completely solublein water.

Molecular weight in the context of a water-soluble polymer, such as PEG,can be expressed as either a number-average molecular weight or aweight-average molecular weight. Unless otherwise indicated, allreferences to molecular weight herein refer to the weight-averagemolecular weight. Both molecular weight determinations, number-averageand weight-average, can be measured using gel permeation chromatographyor other liquid chromatography techniques. Other methods for measuringmolecular weight values can also be used, such as the use of end-groupanalysis or the measurement of colligative properties (e.g.,freezing-point depression, boiling-point elevation, or osmotic pressure)to determine number-average molecular weight or the use of lightscattering techniques, ultracentrifugation or viscometry to determineweight-average molecular weight. The polymers of the invention aretypically polydisperse (i.e., number-average molecular weight andweight-average molecular weight of the polymers are not equal),possessing low polydispersity values of preferably less than about 1.2,more preferably less than about 1.15, still more preferably less thanabout 1.10, yet still more preferably less than about 1.05, and mostpreferably less than about 1.03. As used herein, references will attimes be made to a single water-soluble polymer having either aweight-average molecular weight or number-average molecular weight; suchreferences will be understood to mean that the single-water solublepolymer was obtained from a composition of water-soluble polymers havingthe stated molecular weight.

The terms “active” or “activated” when used in conjunction with aparticular functional group, refer to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “non-reactive” or “inert” group).

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof as well as unprotected forms.

The terms “spacer moiety,” “linkage” or “linker” are used herein torefer to an atom or a collection of atoms used to link interconnectingmoieties such as a terminus of a polymer and an active agent or anelectrophile or nucleophile of an active agent. The spacer moiety may behydrolytically stable or may include a physiologically hydrolyzable orenzymatically degradable linkage.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to15 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl as well as cycloalkylene-containing alkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched. Nonlimiting examples oflower alkyl include methyl, ethyl, n-butyl, i-butyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or Spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8carbon atoms. “Cycloalkylene” refers to a cycloalkyl group that isinserted into an alkyl chain by bonding of the chain at any two carbonsin the cyclic ring system.

“Alkoxy” refers to an —O—R group, wherein R is alkyl, substituted alkyl[preferably C₁₋₆ alkyl (e.g., methoxy, ethoxy, propyloxy, and so forth)or C₁₋₆ substituted alkyl], aryl, substituted aryl,

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenoninterfering substituents, such as, but not limited to: alkyl, C₃₋₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy; lower phenyl;substituted phenyl; nitro, and the like.

“Substituted aryl” is aryl having one or more noninterferingsubstituents. For substitutions on a phenyl ring, the substituents maybe in any orientation (i.e., ortho, meta, or para).

“Noninterfering substituents” are those groups that, when present in amolecule, are typically nonreactive with other functional groupscontained within the molecule.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably sulfur, oxygen, or nitrogen, or a combination thereof.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom that is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from noninterfering substituents.

An “organic radical” as used herein shall include alkyl, substitutedalkyl, aryl and substituted aryl.

“Electrophile” and “electrophilic group” refer to an ion or atom orcollection of atoms, that may be ionic, having an electrophilic center,i.e., a center that is electron seeking, capable of reacting with anucleophile.

“Nucleophile” and “nucleophilic group” refers to an ion or atom orcollection of atoms that may be ionic having a nucleophilic center,i.e., a center that is seeking an electrophilic center or capable ofreacting with an electrophile.

A “physiologically cleavable” or “hydrolyzable” bond is a bond thatreacts with water (i.e., is hydrolyzed) under physiological conditions.Preferred are bonds that have a hydrolysis half-life at pH 8, 25° C. ofless than about 30 minutes. The tendency of a bond to hydrolyze in waterwill depend not only on the general type of linkage connecting two givenatoms but also on the substituents attached to these two given atoms.Appropriate hydrolytically unstable or weak linkages include but are notlimited to carboxylate ester, phosphate ester, anhydrides, acetals,ketals, acyloxyalkyl ether, imine, orthoester, peptide andoligonucleotide.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include, but are not limited to, thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethane, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Pharmaceutically acceptable excipient” refers to an excipient that mayoptionally be included in a composition and that causes no significantadverse toxicological effects to a patient upon administration.

“Therapeutically effective amount” is used herein to mean the amount ofa conjugate that is needed to provide a desired level of the conjugate(or corresponding unconjugated active agent) in the bloodstream or inthe target tissue. The precise amount will depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the therapeutic composition, the intended patientpopulation, the mode of delivery, individual patient considerations, andthe like, and can readily be determined by one skilled in the art.

“Multi-functional” means a polymer having three or more functionalgroups contained therein, where the functional groups may be the same ordifferent. Multi-functional polymeric reagents will typically containfrom about 3-100 functional groups, or from 3-50 functional groups, orfrom 3-25 functional groups, or from 3-15 functional groups, or from 3to 10 functional groups, or will contain 3, 4, 5, 6, 7, 8, 9 or 10functional groups within the polymer backbone.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Substantially” (unless specifically defined for a particular contextelsewhere or the context clearly dictates otherwise) means nearlytotally or completely, for instance, satisfying one or more of thefollowing: greater than 50%, 51% or greater, 75% or greater, 80% orgreater, 90% or greater, and 95% or greater of the condition.

The phrase “substantially nonaqueous” means a composition or reactionmedium: (i) having less than 10,000 parts per million of water (lessthan 1%), more preferably having less than 1,000 parts per million ofwater (less than 0.1%), still more preferably less than 100 parts permillion of water (less than 0.01%), still more preferably less than 10parts per million of water (less than 0.001%). Preferably, but notnecessarily, substantially nonaqueous conditions includes an inertatmosphere.

The term “consumed” as used herein will refer to neutralizing anunreacted activated carbonate reagent [such as di(1-benzotriazolyl)carbonate] using a reactive molecule, which may optionally be a moietybound on a resin or column, to chemically destroy all or most of theunreacted activated carbonate reagent in a chemical process.

“One pot”, as in a one pot synthetic method, means that products from aprevious reaction need not be isolated prior to conducting a subsequentreaction.

Unless the specifically stated to the contrary, the term “combining,” asin “combining” two or more compositions together as part of a syntheticmethod, is not limited with respect to the order of addition.

An “activated carbonate” includes diBTC as well di(1-benzotriazolyl)carbonate (BTC), disuccinimidyl carbonate (“DSC”), p-nitrophenylhaloformate, disuccinimidyl oxalate, and triphosgene even though one ormore of compounds may not be true “carbonates,” (wherein halo isselected from the group consisting of fluoro, chloro, bromo, iodo) andalkoxy-substitutions of one or both of the benzotriazolyl moietieswithin diBTC. An activated carbonate must, however, result in anactivated urethane-, carbonate-, or thiocarbonate-terminated,water-soluble polymer upon reaction with an amine-terminated,hydroxyl-terminated, or thiol-terminated, water-soluble polymer. Anactivated urethane-, carbonate-, or thiocarbonate-terminated,water-soluble polymer, in turn, is a urethane-, carbonate-, orthiocarbonate-terminated, water-soluble polymer that can react witheither a protein or with a reagent to result in a reactivegroup-terminated, water-soluble polymer.

Unless the context clearly dictates otherwise, when the term “about”precedes a numerical value, the numerical value is understood tomean±10% of the stated numerical value.

The Steps of the Synthetic Method

The synthetic method includes the following steps:

(a) combining a composition comprising an amine-terminated orhydroxy-terminated, water-soluble polymer with a composition comprisingan activated carbonate reagent, optionally in the presence of a catalystor acid-neutralizing base, wherein the composition comprising theactivated carbonate reagent is added such that there is an excess of theactivated carbonate reagent relative to the amine-terminated orhydroxy-terminated, water-soluble polymer, to thereby result in acomposition comprising an active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent; and

(b) adding a composition comprising a reactive molecule to thecomposition comprising the active carbonate ester of the water-solublepolymer and unreacted activated carbonate reagent, wherein thecomposition comprising a reactive molecule is added such thatsubstantially all of the unreacted activated carbonate reagent issubstantially consumed.

The combining step results in the formation of, among other things, acomposition comprising an active urethane-, carbonate- orthiol-terminated, water-soluble polymer. A urethane-terminated,water-soluble polymer is formed when a composition comprising anamine-terminated, water-soluble polymer is used. A carbonate-terminated,water-soluble polymer is formed when a composition comprising ahydroxyl-terminated, water-soluble polymer is used. Athiocarbonate-terminated, water-soluble polymer is formed when athiol-terminated, water-soluble polymer is used. Each of these so-formedwater-soluble polymers (i.e., the urethane-terminated, water-solublepolymer, the carbonate-terminated, water-soluble polymer, and thethiocarbonate-terminated, water-soluble polymer) the prepared as part ofthe methods provided herein will be referred to as an active carbonateester of the water-soluble polymer.

The method of the invention is schematically provided below, wherein acomposition comprising the hydroxyl-terminated, water-soluble polymermPEG-OH is used, the activated carbonate reagent is di(1-benzotriazolyl)carbonate, the resulting active carbonate ester is mPEG-BTC, and thereactive molecule is water.

Thus, in an exemplary approach, the method comprises

a) combining a composition comprising a hydroxy-terminated,water-soluble polymer of the following structure: POLY-(X)_(a)—OH,wherein POLY is a water-soluble polymer, X (when present) is an optionalspacer moiety, (a) is either zero or one [preferably, POLY-(X)_(a)-OH isCH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH], with a composition comprisingdi(1-benzotriazolyl) carbonate, wherein the composition comprising thedi(1-benzotriazolyl) carbonate is added such that there is an excess ofthe di(1-benzotriazolyl) relative to the hydroxy-terminated,water-soluble polymer, to thereby result in a composition comprising anactive carbonate ester of a water-soluble polymer having the followingstructure:

wherein POLY, X and (a) are as previously defined, preferably

wherein (n) is an integer from 2 to about 4000, and unreacteddi(1-benzotriazolyl) carbonate (and liberated hydroxybenzotriazole);

b) adding water to the composition comprising the active carbonate esterof the water-soluble polymer and unreacted di(1-benzotriazolyl)carbonate, wherein the water is added such that substantially all of theunreacted activated carbonate reagent is substantially consumed.

The method can advantageously be carried out as a “one-pot” synthesis.In addition, the method does not require a precipitation step to removethe activated carbonate reagent as required in the method disclosed inU.S. Pat. No. 6,624,246. However, if this activated carbonate reagent isthe reagent desired, it may be isolated by precipitation.

Optionally, the method further comprises reacting the active carbonateester of a water soluble polymer (such as mPEG-BTC) in one or morereactions to form a different polymeric reagent. Having formed apolymeric reagent, it is possible to react the polymeric reagent with anactive agent under conjugation conditions to thereby result in awater-soluble polymer-active agent conjugate. While the active carbonateester of the water soluble polymer (such as mPEG-BTC) can be used as anintermediate in the formation of a polymeric reagent, it is alsopossible to use the active carbonate ester (such as mPEG-BTC) itself asa polymeric reagent in a conjugation reaction.

In order to recover active carbonate esters of water-soluble polymers,it is typical to add an excess of a non-solvent. Exemplary non-solventsinclude isopropyl alcohol, diethyl ether, MTBE, heptane, THF, hexane,and combinations thereof.

In one or more embodiments of the invention, the active carbonate esterof the water-soluble polymer will have a structure of the followingformula:POLY-O—(C═O)—O—Rwherein:

POLY is a water-soluble polymer; and

R is an organic radical,

with the proviso that when POLY is a linear, methyl-capped poly(ethyleneglycol), the linear, methyl-capped polyethylene glycol has a molecularweight of at least 103.

Exemplary reactive molecules for use in the method include nucleophilicmolecules such as water, lower alkyl monohydric alcohols (such asmethanol, ethanol, n-propanol, isopropanol, and so forth), lower alkylmonobasic amines, and resins bearing bound reactive carboxylate,hydroxyl, thiol or amine groups (via, for example, an amine gel, such asa Duolite A-7 amine gel). Advantageously, such resin can form part of acolumn through which a composition bearing unreacted activated carbonatereagent can be passed to effect the step of “adding a compositioncomprising a reactive molecule to the composition comprising an activecarbonate ester of the water-soluble polymer and unreacted activatedcarbonate reagent”). It is preferred, however, that the reactivemolecule is water. Water can be added directly. In addition, water canbe added via a moiety that releases water, such a hydrated salt.

In one or more embodiments of the method, the formation of carbondioxide results upon the unreacted activated carbonate reagent beingsubstantially consumed.

The combining step requires, as a composition comprising anamine-terminated, hydroxy-terminated, or thiol-terminated, water-solublepolymer. As used herein, an “amine-terminated, hydroxy-terminated orthiol-terminated, water-soluble polymer” is any water-soluble polymerthat bears at least one amine group (“—NH₂”) or hydroxy group (“—OH”),or thiol group (“—SH”), regardless of whether the amine group, hydroxygroup or thiol group is actually located at a terminus of thewater-soluble polymer. In fact, the amine group (—NH₂) or hydroxy group(“OH”) or thiol group (“—SH”) may be bound to an aromatic group.

The combining step also requires a composition comprising an activatedcarbonate reagent. The activated carbonate reagent use in the method istypically, although not necessarily, selected from the group consistingof di(1-benzotriazolyl) carbonate (BTC), disuccinimidyl carbonate(“DSC”), p-nitrophenyl chloroformate, trichlorophenyl chloroformate,p-nitrophenyl succinimidyl carbonate, p-nitrophenyl 1-benzotriazolylcarbonate, pentafluorophenyl chloroformate, 1,1′-carbonyldiimidazole,disuccinimidyl oxalate, and triphosgene. In certain instances, theactivated carbonate reagent has a structure of one of the followingformulae: R—O(C═O)—OR; R—O(C═O)—OR′; and R—O(C═O)X, wherein R is anorganic radical, R′ is an organic radical different than R, and X is Cl,Br, or I.

The excess of the activated carbonate reagent relative to theamine-terminated or hydroxy-terminated, water-soluble polymer representsone or more of the following: at least about 5 mol % excess, at leastabout 50 mol % excess, at least about a two molar excess; at least abouta four molar excess; and at least about an eight molar excess.

The method for preparing active carbonate esters of water-solublepolymers has utility as, among other things, providing an intermediatethat is useful in the formation of polymeric reagents, as discussed in,for example, U.S. Pat. Nos. 6,624,246, and 5,932,462.

The combining step is typically carried out under substantiallynonaqueous conditions. In addition, aprotic solvent-based compositionsare used in the synthetic methods. In addition, the entire syntheticapproach is often carried out under an inert atmosphere, such as argon.

One or more of the steps of the method are carried out in an organicsolvent. Although any organic solvent can be used and the invention isnot limited in this regard, exemplary organic solvents include thosesolvents selected from the group consisting of halogenated aliphatichydrocarbons, alcohols, aromatic hydrocarbons, halogenated aromatichydrocarbons, ethers, cyclic ethers, and combinations thereof. Examplesof preferred organic solvents include those selected from the groupconsisting of methylene chloride (dichloromethane), chloroform, octanol,toluene, methyl t-butyl ether, tetrahydrofuran, ethyl acetate,diethylcarbonate, acetone, acetonitrile, DMF, DMSO, dimethylacetamide,N-cyclohexylpyrrolidinone, cyclohexane and combinations thereof.

Method for Preparing a Conjugate-Containing Composition

In one or more embodiments of the invention, a method for preparing aconjugate-containing composition is provided, the method comprisingcombining an active agent with an active carbonate ester of awater-soluble polymer (or a polymeric reagent that was prepared usingsuch an active carbonate ester of a water-soluble polymer) to therebyresult in a conjugate-containing composition. Thus, included within theinvention are methods for preparing conjugate-containing compositions.

Conjugate-Containing Compositions

In one or more embodiments of the invention, a conjugate-containingcomposition is provided, the composition resulting from the methodcomprising combining an active agent with an active carbonate ester of awater-soluble polymer (or a polymeric reagent that was prepared usingsuch an active carbonate ester of a water-soluble polymer) as providedherein.

Thus, included within the invention are conjugate-containingcompositions. The compositions (both conjugate compositions and reagentcompositions) are believed to have greater purity than previously knownmethods and also more efficiently prepared.

The Water-Soluble Polymer (“POLY”)

As used herein, the term “water soluble polymer” includes those watersoluble polymers that are biocompatible and nonimmunogenic andspecifically excludes any water soluble polymer segments that are notbiocompatible and nonimmunogenic. With respect to biocompatibility, asubstance is considered biocompatible if the beneficial effectsassociated with use of the substance alone or with another substance(e.g., active agent) in connection with living tissues (e.g.,administration to a patient) outweighs any deleterious effects asevaluated by a clinician, e.g., a physician. With respect tonon-immunogenicity, a substance is considered nonimmunogenic if theintended use of the substance in vivo does not produce an undesiredimmune response (e.g., the formation of antibodies) or, if an immuneresponse is produced, that such a response is not deemed clinicallysignificant or important as evaluated by a clinician. It is particularlypreferred that the water soluble polymer segments described herein aswell as conjugates are biocompatible and nonimmunogenic.

When referring to the polymer, it is to be understood that the polymercan be any of a number of water soluble and non-peptidic polymers, suchas those described herein as suitable for use in the present invention.Preferably, poly(ethylene glycol) (i.e., PEG) is the polymer. The termPEG includes poly(ethylene glycol) in any of a number of geometries orforms, including linear forms, branched or multi-arm forms (e.g., forkedPEG or PEG attached to a polyol core), pendant PEG, or PEG withdegradable linkages therein, to be more fully described below.

The number of functional groups carried by the polymer and the positionof the functional groups may vary. Typically, the polymer will comprise1 to about 25 functional groups, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 functional groups. Linear polymers, such as PEG polymers, willtypically comprise one or two functional groups positioned at theterminus of the polymer chain. If the PEG polymer is monofunctional(i.e., mPEG), the polymer will include a single functional group. If thePEG polymer is difunctional, the polymer may contain two independentlyselected functional groups, one at each terminus of the polymer chain.As would be understood, multi-aim or branched polymers may comprise agreater number of functional groups.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the PEG polymer. Generally speaking, amulti-armed or branched polymer possesses two or more polymer “arms”extending from a central branch point. For example, an exemplarybranched PEG polymer has the structure:

wherein PEG_(i) and PEG₂ are PEG polymers in any of the forms orgeometries described herein, and which can be the same or different, andis a hydrolytically stable linkage. An exemplary branched PEG has thestructure:

wherein poly_(a) and poly_(b) are PEG backbones, such as methoxypoly(ethylene glycol); R″ is a nonreactive moiety, such as H, methyl ora PEG backbone; and P and Q are nonreactive linkages. In a preferredembodiment, the branched PEG polymer is methoxy poly(ethylene glycol)disubstituted lysine. For purposes herein, a multi-arm structure willinclude three or more branches while a branched structure will includeonly two branches.

The branched PEG structure can be attached to a third oligomer orpolymer chain as shown below:

wherein PEG₃ is a third PEG oligomer or polymer chain, which can be thesame or different from PEG₁ and PEG₂.

The PEG polymer can alternatively comprise a forked PEG. Generallyspeaking, a polymer having a forked structure is characterized as havinga polymer chain attached to two or more functional groups via covalentlinkages extending from a hydrolytically stable branch point in thepolymer. An example of a forked PEG is represented by PEG-YCHZ₂, where Yis a linking group and Z is an activated terminal group for covalentattachment to a biologically active agent. The Z group is linked to CHby a chain of atoms of defined length. U.S. Pat. No. 6,362,254, thecontents of which are incorporated by reference herein, disclosesvarious forked PEG structures capable of use in the present invention.The chain of atoms linking the Z functional groups (e.g., hydroxylgroups) to the branching carbon atom serve as a tethering group and maycomprise, for example, an alkyl chain, ether linkage, ester linkage,amide linkage, or combinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups (e.g., hydroxyl groups) covalently attached along the length ofthe PEG backbone rather than at the end of the PEG chain. The pendantreactive groups can be attached to the PEG backbone directly or througha linking moiety, such as an alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more hydrolytically stable or degradable linkagesin the polymer backbone, including any of the above described polymers.For example, PEG can be prepared with ester linkages in the polymerbackbone that are subject to hydrolysis. As shown below, this hydrolysisresults in cleavage of the polymer into fragments of lower molecularweight:-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al., Polymer Preprints, 38(1):582-3 (1997), which isincorporated herein by reference.); phosphate ester linkages formed, forexample, by reacting an alcohol with a phosphate group; hydrazonelinkages which are typically formed by reaction of a hydrazide and analdehyde; acetal linkages that are typically formed by reaction betweenan aldehyde and an alcohol; ortho ester linkages that are, for example,formed by reaction between acid derivatives and an alcohol; andoligonucleotide linkages formed by, for example, a phosphoramiditegroup, e.g., at the end of a polymer, and a 5′ hydroxyl group of anoligonucleotide. The use of many of the above-described degradablelinkages is less preferred due to nucleophilic reactivity of many of theunstable linkages with amine groups.

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms of PEG.

Any of a variety of other polymers comprising other non-peptidic andwater soluble polymer chains can also be used in the present invention.The polymer can be linear, or can be in any of the above-described forms(e.g., branched, forked, and the like). Examples of suitable polymersinclude, but are not limited to, other poly(alkylene glycols),copolymers of ethylene glycol and propylene glycol, poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxyaceticacid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene,polyoxazolines, poly(N-acryloylmorpholine), such as described in U.S.Pat. No. 5,629,384, which is incorporated by reference herein in itsentirety, and copolymers, terpolymers, and mixtures thereof.

Although the molecular weight of the water soluble polymer can varydepending on the desired application, the configuration of the polymerstructure, the degree of branching, and the like, the molecular weightwill satisfy one or more of the following values: greater than 100Daltons; greater than 200 Daltons; greater than 400 Daltons; greaterthan 500 Daltons, greater than 750 Daltons; greater than 900 Daltons;greater than 1,000 Daltons, greater than 1,400 Daltons; greater than1,500 Daltons, greater than 1,900 Daltons; greater than 2,000 Daltons;greater than 2,200 Daltons; greater than 2,500 Daltons; greater than3,000 Daltons; greater than 4,000 Daltons; greater than 4,900 Daltons;greater than 5,000 Daltons; greater than 6,000 Daltons; greater than7,000 Daltons; greater than 7,500 Daltons, greater than 9,000 Daltons;greater than 10,000 Daltons; greater than 11,000 Daltons; greater than14,000 Daltons, greater than 15,000 Daltons; greater than 16,000Daltons; greater than 19,000 Daltons; greater than 20,000 Daltons;greater than 21,000 Daltons; greater than 22,000 Daltons, greater than25,000 Daltons; and greater than 30,000 Daltons. It is understood thatthe maximum limit of molecular weight for any given water solublepolymer segment useful herein is less than about 300,000 Daltons.

The molecular weight of the polymer will typically fall into at leastone of the following ranges: from about 100 Daltons to about 100,000Daltons; from about 200 Daltons to about 60,000 Daltons; from about 300Daltons to about 40,000 Daltons.

Exemplary molecular weights for the water soluble polymer segmentinclude about 100 Daltons, about 200 Daltons, about 300 Daltons, about350 Daltons, about 400 Daltons, about 500 Daltons, about 550 Daltons,about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800Daltons, about 900 Daltons, about 1,000 Daltons, about 2,000 Daltons,about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about4,000 Daltons, about 4,400 Daltons, about 5,000 Daltons, about 6,000Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons,about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000Daltons, about 50,000 Daltons, about 60,000 Daltons, and about 75,000Daltons.

With respect to branched versions of the polymer, exemplary ranges ofsuitable sizes for the total molecular weight of the polymer (as basedessentially on the combined weights of the two water soluble polymerportions) include the following: from about 200 Daltons to about 100,000Daltons; from about 1,000 Daltons to about 80,000 Daltons; from about2,000 Daltons to about 50,000 Daltons; from about 4,000 Daltons to about25,000 Daltons; and from about 10,000 Daltons to about 40,000 Daltons.More particularly, total weight average molecular weight of a branchedversion of the polymer of the invention corresponds to one of thefollowing: 400; 1,000; 1,500; 2,000; 3000; 4,000; 10,000; 15,000;20,000; 30,000; 40,000; 50,000; 60,000; or 80,000.

With respect to PEG, wherein a structure comprising a repeating ethyleneoxide monomer, such as “—(CH₂CH₂O)_(n)—” or “—(OCH₂CH₂)_(n)” can beprovided, preferred values for (n) include: from about 3 to about 3,000;from about 10 to about 3,000; from about 15 to about 3,000; from about20 to about 3,000; from about 25 to about 3,000; from about 30 to about3,000; from about 40 to about 3,000; from about 50 to about 3,000; fromabout 55 to about 3,000; from about 75 to about 3,000; from about 100 toabout 3,000; and from about 225 to about 3,000.

The Spacer Moiety (“X”)

Optionally, a spacer moiety is found in the water-soluble polymers andother structures provided herein. Exemplary spacer moieties include thefollowing: —O—, —S—, —C(O)—, —O—C(O)—, —C(O)—O—, —C(O)—NH—,—NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—, —CH₂—C(O)—O—CH₂—,—CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—,—CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]₀₋₆—(OCH₂CH₂)₀₋₂—, —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —O—C(O)—CH₂—, —O—C(O)—CH₂—CH₂—,—O—C(O)—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,O—C(O)—NH—[CH₂]_(f)—(OCH₂CH₂)_(n)—, and combinations of two or more ofany of the foregoing, wherein (f) is 0 to 6, (n) is 0 to 20 (preferably0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and more preferably4). In addition, each of the foregoing carbon-containing spacer moietiescan have a branched alkyl group attached thereto. Nonlimiting examplesof bivalent cycloalkyl (e.g.; cycloalkylene) groups include C₃₋₈cycloalkyl, such as various isomers of cyclopropadiyl (e.g., 1,1-,cis-11,2-, or trans-1,2-cyclopropylene), cyclobutadiyl, cyclopentadiyl,cyclohexadiyl, and cycloheptadiyl. The cycloalkylene group can besubstituted with one or more alkyl groups, preferably C₁-C₆ alkylgroups.

Having formed the active carbonate ester of a water-soluble polymer, theactive carbonate ester of a water-soluble polymer can be used “as is” asa polymeric reagent useful for a conjugation reaction with an activeagent, or can optionally be further derivatized to form a differentpolymeric reagent bearing a different reactive group. Preferred reactivegroups are selected from the group of electrophiles and nucleophiles.Exemplary reactive groups include hydroxyl (—OH), ester, orthoester,carbonate, acetal, aldehyde, aldehyde hydrate, ketone, vinyl ketone,ketone hydrate, thione, thione hydrate, hemiketal, sulfur-substitutedhemiketal, ketal, alkenyl, acrylate, methacrylate, acrylamide, sulfone,amine, hydrazide, thiol, disulfide, thiol hydrate, carboxylic acid,isocyanate, isothiocyanate, maleimide

succinimide

benzotriazole

vinylsulfone, chloroethylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates,thiosulfonate, tresylate, and silane. Specific examples of preferredreactive groups include amine, carboxylic acid, ester, aldehyde, acetal,succinimide, and maleimide.

Illustrative examples of spacer moieties and reactive group combinationsinclude

wherein r is 1-5, r′ is 0-5, and R₇ is aryl or alkyl.

For example, the active carbonate ester of a water-soluble polymer canbe reacted with amino acids to form amino acid derivatives. Thus, forexample, mPEG-BTC esters can be reacted with lysine to form a polymericlysine derivative. For example, one such lysine [i.e.,H₂N(CH₂)₄CH(NH₂)CO₂H.HCl] derivative is a doubly PEGylated lysine,wherein the two PEGs are linked to the lysine amines by carbamate bonds,as shown below (wherein n is 2 to about 4000).

Such PEG derivatives of lysine are useful as reagents for preparation ofPEG derivatives of proteins. These PEG derivatives often offeradvantages over non-PEGylated proteins, such as longer circulatinglife-times in vivo, reduced rates of proteolysis, and loweredimmunogenicity. In another aspect, PEG BTC derivatives are used directlyin attaching PEG to proteins through carbamate linkages and may offeradvantages similar to those described for the lysine PEG derivatives.

Thus for example, the above “PEG2 Acid” can be reacted withdicyclohexylcarbodiimide and N-hydroxysuccinimide to form a PEG2 activeester (N-hydroxysuccinimide) of the PEG2 acid. Then, in a subsequentreaction, the PEG2 active ester can be reacted, in the presence of atertiary amine base, with a trifluoroacetic acid salt of the ethylenediamine monoamide of 3-N-maleimidopropanoic acid to form a polymericreagent bearing a maleimide group capable of reacting with thiol groupson proteins, bioengineered biomolecules and other therapeutic moleculesof interest.

Polymeric reagent bearing a maleimide group (wherein n is an integerfrom 2 to 4000)

By reacting the above polymeric reagent bearing a maleimide group with athiol-containing active agent [“(Active Agent)-HS”, such as acysteine-containing polypeptide, protein or other thiol-containingbiomolecule] under conjugation reaction conditions, a conjugate havingthe following structure is formed

conjugate [wherein n is an integer from 2 to 4000 and “S-(Active Agent)is a residue of the thiol-containing active agent]

Biologically Active Conjugates

For any given polymer, the methods described above advantageouslyprovide the ability to further transform the polymer (either prior orsubsequent to any deprotection step) so that it bears a specificreactive group. Thus, using techniques well known in the art, thepolymer can be functionalized to include a reactive group (e.g., activeester, thiol, maleimide, aldehyde, ketone, and so forth).

For example, when the polymer bears a carboxylic acid as the reactivegroup, the corresponding ester can be formed using conventionaltechniques. For example, the carboxylic acid can undergo acid-catalyzedcondensation with an alcohol, thereby providing the corresponding ester.One approach to accomplish this is to use the method commonly referredto as a Fischer esterification reaction. Other techniques for forming adesired ester are known by those of ordinary skill in the art.

For example, when the polymer bears a carboxylic acid as the reactivegroup, the corresponding ester can be formed using conventionaltechniques. For example, the carboxylic acid can undergo acid-catalyzedcondensation with an alcohol, thereby providing the corresponding ester.One approach to accomplish this is to use the method commonly referredto as a Fischer esterification reaction. Other techniques for forming adesired ester are known by those of ordinary skill in the art.

In addition, polymers bearing a carboxylic acid can be modified to formuseful reactive groups other than esters. For example, the carboxylicacid can be further derivatized to form acyl halides, acylpseudohalides, such as acyl cyanide, acyl isocyanate, and acyl azide,neutral salts, such as alkali metal or alkaline-earth metal salts (e.g.calcium, sodium, and barium salts), esters, anhydrides, amides, imides,hydrazides, and the like. In a preferred embodiment, the carboxylic acidis esterified to form an N-succinimidyl ester, o-, m-, or p-nitrophenylester, 1-benzotriazolyl ester, imidazolyl ester, or N-sulfosuccinimidylester. For example, the carboxylic acid can be converted into thecorresponding N-succinimidyl ester by reacting the carboxylic acid withdicyclohexyl carbodiimide (DCC) or diisopropyl carbodiimide (DIC) in thepresence of a N-hydroxysuccinimide.

The steps of the synthesis methods described above take place in anappropriate solvent. One of ordinary skill in the art can determinewhether any specific solvent is appropriate for any given reaction.Typically, however, the solvent is a nonpolar solvent or a polar aproticsolvent. Nonlimiting examples of nonpolar solvents include benzene,xylene, dioxane, tetrahydrofuran (THF), and toluene. Exemplary polaraprotic solvents include, but are not limited to, acetonitrile, DMSO(dimethyl sulfoxide), HMPA (hexamethylphosphoramide), DMF(dimethylformamide), DMA (dimethylacetamide), and NMP(N-methylpyrrolidinone).

The method of preparing the polymers optionally comprises an additionalstep of isolating and recovering the polymer once it is formed. Knownmethods can be used to isolate the polymer, but it is particularlypreferred to use chromatography, e.g., size exclusion chromatography.Alternately or in addition, the method includes the step of purifyingthe polymer once it is formed. Again, standard art-known purificationmethods can be used to purify the polymer. Isolation of the activecarbonate ester of a water-soluble polymer can also be accomplished bydistilling off the solvent using art-known methods.

The polymers of the invention can be stored under an inert atmosphere,such as under argon or under nitrogen. In this way, potentiallydegradative processes associated with, for example, atmospheric oxygen,are reduced or avoided entirely. In some cases, to avoid oxidativedegradation, antioxidants, such as butylated hydroxyl toluene (BHT), canbe added to the final product prior to storage. In addition, it ispreferred to minimize the amount of moisture associated with the storageconditions to reduce potentially damaging reactions associated withwater. Moreover, it is preferred to keep the storage conditions dark inorder to prevent certain degradative processes that involve light. Thus,preferred storage conditions include one or more of the following:storage under dry argon or another dry inert gas; storage attemperatures below about −15° C.; storage in the absence of light; andstorage with a suitable amount (e.g., about 50 to about 500 parts permillion) of an antioxidant such as BHT.

The above-described polymers are useful for conjugation to biologicallyactive agents or surfaces comprising at least one group suitable forreaction with the reactive group on the polymer. For example, aminogroups (e.g., primary amines), hydrazines, hydrazides, and alcohols onan active agent or surface will react with a carboxylic acid group onthe polymer. In addition, a more “activated” version of the carboxylicacid of the polymer can be prepared in order to enhance reactivity tothe biologically active agent or surface. Methods for activatingcarboxylic acids are known in the art and include, for example, themethod for forming an active ester described above. Other approaches foractivating a carboxylic acid are known to those of ordinary skill in theart.

Typically, the polymer is added to the active agent or surface at anequimolar amount (with respect to the desired number of groups suitablefor reaction with the reactive group) or at a molar excess. For example,the polymer can be added to the target active agent at a molar ratio ofabout 1:1 (polymer:active agent), 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1,or 10:1. The conjugation reaction is allowed to proceed untilsubstantially no further conjugation occurs, which can generally bedetermined by monitoring the progress of the reaction over time.Progress of the reaction can be monitored by withdrawing aliquots fromthe reaction mixture at various time points and analyzing the reactionmixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitableanalytical method. Once a plateau is reached with respect to the amountof conjugate formed or the amount of unconjugated polymer remaining, thereaction is assumed to be complete. Typically, the conjugation reactiontakes anywhere from minutes to several hours (e.g., from 5 minutes to 24hours or more). The resulting product mixture is preferably, but notnecessarily, purified to separate out excess reagents, unconjugatedreactants (e.g., active agent), undesired multi-conjugated species, andfree or unreacted polymer. The resulting conjugates can then be furthercharacterized using analytical methods such as MALDI, capillaryelectrophoresis, gel electrophoresis, and/or chromatography.

With respect to polymer-active agent conjugates, the conjugates can bepurified to obtain/isolate different conjugated species. Alternatively,and more preferably for lower molecular weight (e.g., less than about 20kiloDaltons, more preferably less than about 10 kiloDaltons) polymers,the product mixture can be purified to obtain the distribution ofwater-soluble polymer segments per active agent. For example, theproduct mixture can be purified to obtain an average of anywhere fromone to five PEGs per active agent (e.g., protein), typically an averageof about 3 PEGs per active agent (e.g., protein). The strategy forpurification of the final conjugate reaction mixture will depend upon anumber of factors, including, for example, the molecular weight of thepolymer employed, the particular active agent, the desired dosingregimen, and the residual activity and in vivo properties of theindividual conjugate(s).

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography. That is to say, gelfiltration chromatography is used to fractionate differently numberedpolymer-to-active agent ratios (e.g., 1-mer, 2-mer, 3-mer, and so forth,wherein “1-mer” indicates 1 polymer to active agent, “2-mer” indicatestwo polymers to active agent, and so on) on the basis of their differingmolecular weights (where the difference corresponds essentially to theaverage molecular weight of the water-soluble polymer segments). Forexample, in an exemplary reaction where a 100 kDa protein is randomlyconjugated to a PEG alkanoic acid having a molecular weight of about 20kDa, the resulting reaction mixture will likely contain unmodifiedprotein (MW 100 kDa), mono-pegylated protein (MW 120 kDa), di-pegylatedprotein (MW 140 kDa), and so forth. While this approach can be used toseparate PEG and other polymer conjugates having different molecularweights, this approach is generally ineffective for separatingpositional isomers having different polymer attachment sites within theprotein. For example, gel filtration chromatography can be used toseparate mixtures of PEG 1-mers, 2-mers, 3-mers, and so forth, althougheach of the recovered PEG-mer compositions may contain PEGs attached todifferent reactive amino groups (e.g., lysine residues) within theactive agent.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, (iii) iodine testing for PEG content [Sims et al. (1980) Anal.Biochem, 107:60-63], and (iv) sodium dodecyl sulfphate polyacrylamidegel electrophoresis (SDS PAGE), followed by staining with barium iodide.

Separation of positional isomers is carried out by reverse phasechromatography using a reverse phase-high performance liquidchromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac) orby ion exchange chromatography using an ion exchange column, e.g., aSepharose™ ion exchange column available from Amersham Biosciences.Either approach can be used to separate polymer-active agent isomershaving the same molecular weight (positional isomers).

Following conjugation, and optionally additional separation steps, theconjugate mixture can be concentrated, sterile filtered, and stored atlow a temperature, typically from about −20° C. to about −80° C.Alternatively, the conjugate may be lyophilized, either with or withoutresidual buffer and stored as a lyophilized powder. In some instances,it is preferable to exchange a buffer used for conjugation, such assodium acetate, for a volatile buffer such as ammonium carbonate orammonium acetate, that can be readily removed during lyophilization, sothat the lyophilized powder is absent residual buffer. Alternatively, abuffer exchange step may be used using a formulation buffer, so that thelyophilized conjugate is in a form suitable for reconstitution into aformulation buffer and ultimately for administration to a mammal.

The polymers of the invention can be attached, either covalently ornoncovalently, to a number of entities including films, chemicalseparation and purification surfaces, solid supports, metal surfacessuch as gold, titanium, tantalum, niobium, aluminum, steel, and theiroxides, silicon oxide, macromolecules (e.g., proteins, polypeptides, andso forth), and small molecules. Additionally, the polymers can also beused in biochemical sensors, bioelectronic switches, and gates. Thepolymers can also be employed as carriers for peptide synthesis, for thepreparation of polymer-coated surfaces and polymer grafts, to preparepolymer-ligand conjugates for affinity partitioning, to preparecross-linked or non-cross-linked hydrogels, and to preparepolymer-cofactor adducts for bioreactors.

A biologically active agent for use in coupling to a polymer aspresented herein may be any one or more of the following. hypnotics andsedatives, psychic energizers, tranquilizers, respiratory drugs,anticonvulsants, muscle relaxants, antiparkinson agents (dopamineantagonists), analgesics, anti-inflammatories, antianxiety drugs(anxiolytics), appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxicants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

Examples of active agents suitable for use in covalent attachment to thereactive polymer of the invention include, but are not limited to,calcitonin, erythropoietin (EPO), Factor VIII, Factor IX, ceredase,cerezyme, cyclosporin, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), growthhormone, human growth hormone (HGH), growth hormone releasing hormone(GHRH), heparin, low molecular weight heparin (LMWH), interferon alpha,interferon beta, interferon gamma, interleukin-1 receptor,interleukin-2, interleukin-1 receptor antagonist, interleukin-3,interleukin-4, interleukin-6, luteinizing hormone releasing hormone(LHRH), factor IX insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), insulin-like growthfactor (IGF), insulintropin, macrophage colony stimulating factor(M-CSF), nerve growth factor (NGF), tissue growth factors, keratinocytegrowth factor (KGF), glial growth factor (GGF), tumor necrosis factor(TNF), endothelial growth factors, parathyroid hormone (PTH),glucagon-like peptide thymosin alpha 1, IIb/IIIa inhibitor, alpha-1antitrypsin, phosphodiesterase (PDE) compounds, VLA-4 inhibitors,bisphosphonates, respiratory syncytial virus antibody, cystic fibrosistransmembrane regulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), anti-CMV antibody,13-cis retinoic acid, macrolides such as erythromycin, oleandomycin,troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin,flurithromycin, dirithromycin, josamycin, spiromycin, midecamycin,leucomycin, miocamycin, rokitamycin, andazithromycin, and swinolide A;fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin,pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin,irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin,aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin,amikacin, kanamycin, neomycin, and streptomycin, vancomycin,teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin,colistimethate, polymixins such as polymixin B, capreomycin, bacitracin,penems; penicillins including penicllinase-sensitive agents likepenicillin G, penicillin V, penicllinase-resistant agents likemethicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin,nafcillin; gram negative microorganism active agents like ampicillin,amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonalpenicillins like carbenicillin, ticarcillin, azlocillin, mezlocillin,and piperacillin; cephalosporins like cefpodoxime, cefprozil, ceftbuten,ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin,cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cefaclor,cefadroxil, cephaloglycin, cefuroxime, ceforanide, cefotaxime,cefatrizine, cephacetrile, cefepime, cefixime, cefonicid, cefoperazone,cefotetan, cefinetazole, ceftazidime, loracarbef, and moxalactam,monobactams like aztreonam; and carbapenems such as imipenem, meropenem,pentamidine isethiouate, albuterol sulfate, lidocaine, metaproterenolsulfate, beclomethasone diprepionate, triamcinolone acetamide,budesonide acetonide, fluticasone, ipratropium bromide, flunisolide,cromolyn sodium, ergotamine tartrate and where applicable, analogues,agonists, antagonists, inhibitors, and pharmaceutically acceptable saltforms of the above. In reference to peptides and proteins, the inventionis intended to encompass synthetic, native, glycosylated,unglycosylated, pegylated forms, and biologically active fragments andanalogs thereof.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation can also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant can be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases can be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The pharmaceutical preparations encompass all types of formulations andin particular those that are suited for injection, e.g., powders thatcan be reconstituted as well as suspensions and solutions. The amount ofthe conjugate (i.e., the conjugate formed between the active agent andthe polymer described herein) in the composition will vary depending ona number of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container (e.g., avial). In addition, the pharmaceutical preparation can be housed in asyringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining stability and other parameters of thecomposition, and then determining the range at which optimal performanceis attained with no significant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

As previously described, the conjugates can be administered parenterallyby intravenous injection, or less preferably by intramuscular or bysubcutaneous injection. Suitable formulation types for parenteraladministration include ready-for-injection solutions, dry powders forcombination with a solvent prior to use, suspensions ready forinjection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with a conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The method of administering may be used to treat anycondition that can be remedied or prevented by administration of theparticular conjugate. Those of ordinary skill in the art appreciatewhich conditions a specific conjugate can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the patient as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature. Generally, a therapeutically effectiveamount will range from about 0.001 mg to 100 mg, preferably in dosesfrom 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXPERIMENTAL

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis, biochemistry, proteinpurification and the like, which are within the skill of the art. Suchtechniques are fully explained in the literature. See, for example, J.March, Advanced Organic Chemistry: Reactions Mechanisms and Structure,4th Ed. (New York: Wiley-Interscience, 1992), supra.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. and pressure is at ornear atmospheric pressure at sea level. Each of the following examplesis considered to be instructive to one of ordinary skill in the art forcarrying out one or more of the embodiments described herein.

Example 1 Preparation of mPEG(5,000 Da)-BTC

A solution of mPEG(5,000 Da)-OH (MW 5,000, 15 g, 0.003 moles),di(1-benzotriazolyl) carbonate (4.0 g of 70% mixture, 0.000945 moles),and pyridine (2.2 ml) in acetonitrile (30 ml) was stirred at roomtemperature under nitrogen overnight. The NMR analysis showed that theobtained mPEG(5,000 Da)-BTC was 100% substituted and the reactionmixture contained 0.0012 moles of residual di(1-benzotriazolyl)carbonate. A calculated amount of distilled water (0.025 g, calculatedto provide an excess amount relative to diBTC) was added and the mixturewas stirred for three hours. Consecutive NMR analysis showed thatmPEG(5,000 Da)-BTC was still 100% substituted but residualdi(1-benzotriazolyl) carbonate was completely hydrolyzed. The solventwas removed by distillation; the residue was dried under vacuum at roomtemperature to yield 14.8 g of product which was shown by ¹H NMRanalysis to be 100% substituted. Expected ¹H NMR (d₆-DMSO): 3.23 ppm (s,—OCH₃, 3H), 3.51 ppm (s, polymer backbone), 4.62 ppm (m,mPEG-O—CH₂—OCO₂—, 2H), 7.41-8.21 ppm (complex mult, benzotriazoleprotons, 4H).

Example 2 Preparation of mPEG(20,000 Da)-BTC

A solution of mPEG(20,000 Da)-OH (MW 20,000, 20 g, 0.001 moles),di(1-benzotriazolyl) carbonate (3.4 g of 70% mixture, 0.00803 moles),and pyridine (3.0 ml) in acetonitrile (40 ml) was stirred at roomtemperature under nitrogen overnight. The NMR analysis showed that theobtained mPEG(20,000 Da)-BTC was 100% substituted and the reactionmixture contained 0.0027 moles of residual di(1-benzotriazolyl)carbonate. A calculated amount of distilled water (0.050 g, calculatedto provide an excess amount relative to diBTC) was added and the mixturewas stirred for three hours. Consecutive NMR analysis showed thatmPEG(20,000 Da) BTC was still 100% substituted but residualdi(1-benzotriazolyl) carbonate was completely hydrolyzed. The obtainedmixture was used directly to prepare mPEG(20,000 Da)-amine. ¹H NMR(d₆-DMSO): 3.23 ppm (s, —OCH₃, 3H), 3.51 ppm (s, polymer backbone), 4.62ppm (m, mPEG-O—CH₂—OCO₂—, 2H), 7.41-8.21 ppm (complex mult,benzotriazole protons, 4H).

Example 3 Preparation of mPEG(20,000 Da)-Amine from mPEG(20,000 Da)-BTC

The solution of mPEG(20,000 Da)-benzotriazole carbonate (20.0 g, 0.001moles) in acetonitrile (40 ml) prepared in Example 2, is added dropwiseto the solution of 2,2′-(ethylenedioxy)bis(ethylamine) (Sigma-Aldrich;FW=148.21, 3 g, 0.020 moles) in acetonitrile (40 ml) and the reactionmixture is stirred for two hours at room temperature under argonatmosphere. Next the solvent is evaporated to dryness. The crude productis dissolved in methylene chloride and precipitated with isopropylalcohol. The wet product is dried under reduced pressure. Yield 18.2 g.Expected ¹H NMR (d₆-DMSO): 2.64 ppm (t, —CH₂-amine-, 2H), 3.11 ppm (q,—CH₂ —NH—,), 3.23 ppm (s, —OCH₃, 3H), 3.51 ppm (s, PEG backbone), 4.04ppm (m, —CH₂—O(C═O)—, 2H), 7.11 ppm (t, —(C═O)—NH—, 1H). Cation exchangechromatography: mPEG(20,000 Da)-Amine 97.7%.

Example 4 Preparation of mPEG(20,000 Da)-Succinimidyl Carbonate

A solution of mPEG(20,000 Da)-OH (MW 20,000, 60 g, 0.003 moles),N,N′-disuccinimidyl carbonate (2.4 g, 0.00937 moles), and pyridine (6.5ml) in acetonitrile (300 ml) was stirred at room temperature undernitrogen overnight. The NMR analysis showed that the obtainedmPEG(20,000 Da)-succinimidyl carbonate was 100% substituted and thereaction mixture contained 0.0034 moles of residual N,N′-disuccinimidylcarbonate. A calculated amount of distilled water (0.097 g, calculatedto provide an excess amount relative to diBTC) was added and the mixturewas stirred for four hours. Consecutive NMR analysis showed thatmPEG(20,000 Da)-succinimidyl carbonate was still 100% substituted butresidual N,N′-disuccinimidyl carbonate was completely hydrolyzed. Thesolvent was removed by distillation; the residue was dried under vacuumto yield 56.8 g of product which was shown by ¹H NMR to be 100%substituted. ¹H NMR (d₆-DMSO): 2.80 ppm (s, succinimide, 4H), 3.23 ppm(s, —OCH₃, 3H), 3.51 ppm (s, polymer backbone), 4.62 ppm (m,mPEG-O—CH₂—OCO₂—, 2H).

Example 5 Preparation of mPEG(20,000 Da)-NPC

A solution of mPEG(20,000 Da)-OH (MW 20,000 Da, 60 g, 0.003 moles),p-nitrophenyl chloroformate (NPC) (1.9 g, 0.00943 moles), and pyridine(6.5 ml) in acetonitrile (300 ml) was stirred at room temperature undernitrogen overnight. The NMR analysis showed that the obtainedmPEG(20,000 Da)-NPC was 100% substituted and the reaction mixturecontained 0.0034 moles of residual p-nitrophenyl chloroformate. Thereaction mixture was passed through gel column (Duolite A-7 amine gel)that allows circulation of small molecules but largely excludespolymeric species. Consecutive NMR analysis showed that mPEG(20,000Da)-NPC was still 100% substituted but no residual p-nitrophenylchloroformate. ¹H NMR (CDCl₃): 3.39 ppm (s, —OCH₃, 3H), 3.58 ppm (s,polymer backbone), 4.37 ppm (m, mPEG-O—CH₂—OCO₂—, 2H), 7.44 ppm (m,aromatic protons, 2H), 8.27 ppm (m, aromatic protons, 2H).

Example 6 Preparation of mPEG(22,000 Da)-Propionic Acid from mPEG(20,000Da)-NPC

To a solution of mPEG(20,000 Da)-NPC (60.0 g, 0.0030 moles from Example5) in acetonitrile, PEG(2,000 Da)-α-amino-w-propionic acid (6.6 g,0.0033 moles) and triethylamine (1.8 ml) were added and the reactionmixture was stirred for six hours at room temperature under argonatmosphere. Next, the mixture was filtered and solvent was evaporated todryness. The crude product was dissolved in methylene chloride andprecipitated with isopropyl alcohol. The wet product was dried underreduced pressure. Yield 54.3 g. ¹H NMR (d₆-DMSO): 2.44 ppm (t,—CH₂—COO—, 2H), 3.11 ppm (q, —CH₂ —NH—, 2H), 3.24 ppm (s, —OCH₃), 3.51ppm (s, PEG backbone), 4.04 ppm (m, —CH₂—O(C═O)—, 2H), 7.11 ppm (t,—(C═O)—NH—, 1H). Anion exchange chromatography: M-PEG(20,000Da)-Propionic Acid 96.5%.

Example 7 Preparation of mPEG(20,000 Da)-BTC Large Scale Synthesis

mPEG(20,000 Da)-OH (28 kg) was dissolved in anhydrous acetonitrile (93kg) under argon atmosphere. The mixture was dried by passing through amolecular sieves cartridge, then the cartridge was washed with 50 L ofacetonitrile. The drying process was repeated until the water contentwas less than 100 ppm. Di(1-benzotriazolyl) carbonate (1285 g of 42%mixture) was then combined with about 2000 mL of acetonitrile in aseparate vessel to form a slurry, which was added under argon to thepreviously prepared mixture. The separate vessel was rinsed twice withacetonitrile (about 200 mL) to enhance quantitative transfer. Pyridine(200 mL) was then added under argon and followed by mixing at 31° C.±3°C. for 12 hours. Water (about 20 mL), diluted with acetonitrile (1 L),was added and the entire reaction mixture was mixed at 31° C.±3° C. for60 minutes. Thereafter, the reaction mixture was distilled under reducedpressure and the product was collected and allowed to cool. Isopropanol(551 kg) with a low water content was then added. After addition of theisopropanol was complete, the resulting precipitate was mixed for aminimum of thirty minutes. Thereafter, the precipitate was isolated andwashed with isopropanol (about 90 kg) under argon. Finally, theprecipitate was dried under vacuum. The percent yield and total amountof product was 22 kg (79% yield). NMR analysis showed that the productcontained 98.6% of desired mPEG(20,000 Da)-BTC and 1.4% of startingmPEG(20,000 Da)-OH. No isopropyl benzotriazole carbonate was detected.

Comparative Example 8 Preparation of mPEG(20,000 Da)-BTC Large ScaleSynthesis with No Water Treatment

The large scale synthesis of mPEG(20,000 Da)-BTC described in theExample 7 was repeated but water was not added to the reaction mixtureto hydrolyze residual di(1-benzotriazolyl) carbonate. NMR analysisshowed that the final product contained 44 mol % of isopropylbenzotriazolyl carbonate. Thus, this experiment shows that large scaleproduction with the need to isolate and remove activated carbonatereagents is possible. However, loss on precipitation is predicted to be10-20% based on comparative experiments.

1. A synthetic method comprising: (a) combining a composition comprisingan aprotic solvent and a hydroxy-terminated, water-soluble polymerhaving the following structure:CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH wherein (n) is an integer from 2 to about4000, with a composition comprising di(1-benzotriazolyl) carbonate,wherein the composition comprising the di(1-benzotriazolyl) carbonate isadded such that there is an excess of the di(1-benzotriazolyl) carbonaterelative to the hydroxy-terminated, water-soluble polymer, to therebyresult in a composition comprising an active carbonate ester of thewater-soluble polymer having the following structure:

wherein (n) is an integer from 2 to about 4000, and unreacteddi(1-benzotriazolyl) carbonate; (b) adding a composition comprising areactive molecule selected from the group consisting of water, a loweralkyl monohydric alcohol, hydrogen sulfate and a lower alkyl monobasicamine to the composition comprising the active carbonate ester of awater-soluble polymer and unreacted di(1-benzotriazolyl) carbonate,wherein the composition comprising the reactive compound is added suchthat substantially all of the unreacted di(1-benzotriazolyl) carbonateis substantially consumed; and (c) reacting the active carbonate esterof a water-soluble polymer lysine to form a polymeric lysine derivative.2. The method as in claim 1, wherein the reactive molecule is selectedfrom the group consisting of water, a lower alkyl monohydric alcohol,hydrogen sulfide, and a lower alkyl monobasic amine.
 3. The method as inclaim 1, wherein the reactive molecule is selected from hydroxyalkylattached to a solid support, mercaptoalkyl attached to a solid support,primary amine attached to a solid support, and secondary amine attachedto a secondary support.
 4. The method as in claim 1, wherein thepolymeric lysine derivative has the following structure:

wherein each (n) is an integer from 2 to about 4000.